Objective The Cap protein of Eel circovirus (EeCV) was prepared using a prokaryotic expression system, with a view to laying the foundation for the preparation of EeCV subunit vaccine and related biological products.

Method The EeCV capsid protein genome sequence (GenBank accession number: NC_023421.1) was used as a reference for codon optimization and plasmid synthesis, and the cap protein gene was amplified by designing specific primers. The target gene was cloned into pET-32a vector to construct pET32a-EeCV-Cap recombinant expression plasmid. The EeCV Cap recombinant protein was prepared by transforming the recombinant plasmid into the expression host bacterium BL21 (DE3) followed by induction with IPTG. Western blot was performed to confirm expression of the recombinant Cap protein.

Result The results showed that the recombinant pET32a-EeCV-Cap was mainly expressed as a soluble protein in E. coli, and a purified target protein was obtained using nickel affinity chromatography, with the highest expression levels achieved at a final IPTG concentration of 0.5 mM and induction at 20°C for 16 hours. Western blot analysis showed that a single band of 31 kDa was detected, which was consistent with the calculated molecular weight. The negative phosphotungstic acid staining of the protein suspension showed a large number of regular VLPs with diameters of 20 nm under transmission electron microscope.

Conclusion In conclusion, this study achieved the soluble expression of EeCV Cap protein in E. coli, and a single target protein was obtained using modified nickel affinity chromatography, which spontaneously assemble into virus-like particles in vitro. These VLPs have potential application in development of subunit vaccines and related biological products.